Sterol 14?-demethylase (CYP51) is the most widely distributed and perhaps the oldest of the > 13,000 P450s known to date. This monooxygenase catalyzes a unique three step reaction (14?-methyl ->14?- alcohol->14?-aldehyde->14?-demethylated product plus formic acid) removing the 14?-methyl group from the initial cyclized intermediate in sterol biosynthesis, i.e. lanosterol in cholesterol biosynthesis. CYP51 is a drug target in eukaryotic pathogens because the reaction it catalyzes is essential for membrane formation and therefore loss of this activity is lethal. It has been studied extensively as a drug target in yeast and filamentous fungi where azoles that bind to the CYP heme iron have been found to be very effective drugs. Other eukaryotic human pathogens have not been studied in great detail and we have begun a detailed analysis of CYP51 in trypanosomes and leishmania, including whether it can serve as a drug target for killing these organisms which cause more than one hundred million deaths each year in the 'third world' and is becoming a serious global problem, mainly due to human migration and growing immunodeficiency. Our detailed investigation of CYP51 from Trypanosomatidae has been supported during the first two funding cycles of this grant, and during this current cycle we have also characterized novel chemical scaffolds which we believe are the basis of very efficient inhibitors of CYP51 from protozoa. This competing renewal application consists of three Specific Aims. First, is structure-based development of selective inhibitors for protozoan CYP51s. Here we will synthesize derivatives of the three original scaffolds we discovered (azole, pyridine and substrate based) and analyze the best of these derivatives in four test systems: protozoa themselves, human cells, human cells infected with protozoa and mouse models for Chagas disease. Second, we will test these scaffolds and their derivatives as potential drugs for treatment of infection by Candida albicans and Aspergillus (A) fumigatus and A. flavus. These fungi are highly pathogenic in humans, especially in immunocompromised patients, and after characterization by biochemistry and biophysics (including X-ray structure) of their CYP51s we will establish which scoffids and derivatives will be most effective for antifungal drug design. Third, we will compare structure-function characteristics of CYP51s from three different biological kingdoms (protozoa/fungi/human) to establish in detail the basis on which we can identify what features of both the enzymes and the inhibitors will lead to rational design of pathogen-selective drugs and drugs effective for CYP51-related azole resistance. Overall, the results arising from these studies will direct future approaches for drug development which will have major importance in global health. PUBLIC HEALTH RELEVANCE: Sterol 14?-demethylases are major drug targets for treatment of fungal infections and are emerging targets for human trypanosomiasis and leishmaniasis. We have identified three novel CYP51 inhibitory scaffolds highly effective for Trypanosomatidae and determined crystal structures of CYP51s from three Trypanosomatidae pathogens (T. cruzi, T. brucei and L. infantum) in complexes with the inhibitors. This project is aimed at further development of our studies by determining X-ray structures of CYP51s from pathogenic fungi (Aspergillus fumigatus and Candida albicans) and use of the combined structure/function information for CYP51 structure- directed modifications of our new scaffolds into even better, pathogen-specific inhibitors which are also effective against drug-resistance.